The present invention relates to mammals engineered to have a functional deficiency in the LKB1 gene as well as to a preparation method thereof. The human LKB1 gene is a causative gene of Peutz-Jeghers syndrome, and thus such mammals can be used to develop methods to treat the disease and therapeutic agents therefor.
Peutz-Jeghers syndrome (MIM 175200, PJS) is a human genetic disease the major go symptoms of which include polyposis in the digestive tract and pigmental spot formation on mucous membranes and skin. PJS is inherited in an autosomal-dominant fashion. In 1997, Hemminki et al. reported that the causative gene for the disease was mapped on p13.3 region of chromosome 19 based on the linkage analysis of PJS patient families (Hemminki et al., Nat. Genet. 15:87-90, 1997). There exists the novel serine/threonine kinase, LKB1, which was found by the present inventors in this region. Based on the fact, Jenne et al. predicted that this gene is a candidate for the causative gene and carried out mutational analysis of the LKB1 (STK11) gene in PJS patients. Their results showed that all the patients tested had mutations in the LKB1 gene (PCT/JP98-05357; Jenne et al., Nat. Genet. 18:38-43, 1998). In addition, other groups also reported similar results, one after another. More than 60 types of mutations in the LKB1 gene have been found to date in PJS patients (Hemminki et al., Nature 391:184-7, 1998; Nakagawa et al., Hum. Genet. 103:168-72, 1998; Resta et al., Cancer Res. 58:4799-801, 1998; Ylikorkala et al., Hum. Mol. Genet. 8:45-51, 1999).
Further, the present inventors have demonstrated that the product of the LKB1 gene is a kinase having the ability of autophosphorylation, and that missense mutations that have been found in PJS patients result in loss of the kinase activity (Mehenni et al., Am. J. Hum. Genet. 63:1641-50, 1998).
Based on these findings, it has been clarified that functional deficiency of the LKB1 serine/threonine kinase due to gene mutations is the cause of PJS.
Further, epidemiological studies have shown that PJS patients have markedly increased risks for the onset of a variety of cancers compared with healthy normal persons. Thus, it has been suggested that the PJS causative gene should have a tumor suppressor gene like activity. In fact, it has been reported that mutations are found in the LKB1 gene in some sporadic cancers unrelated to PJS. Therefore, it has been elucidated that functional inactivation of the LKB1 gene is related to general sporadic cancers (Dong et al., Cancer Res. 58:3787-90, 1998; Rowan et al., J. Invest. Dermatol. 112:509-11, 1999; Guldberg et al., Oncogene 18:1777-80, 1999). However, specific physiological functions of LKB1 in normal cells as well as the mechanism for polyposis or cancerization induced by its functional inactivation has remained obscure.
This situation led to the present invention, and the object of the present invention is to provide non-human mammals useful for analyzing LKB1 functions and for developing agents to treat diseases caused by LKB1 mutations. More specifically, the object of the invention is to provide knockout animals, in which the expression of the LKB1 gene is artificially suppressed, as well as to provide methods for preparing the animals. In a preferred embodiment, the present invention provides non-human mammals in which deletion of the endogenous LKB1 is achieved in an inducible manner.
The present inventors created mammal models, in which the LKB1 gene is artificially deleted, or in which the deletion can be induced. Specifically, as shown in the Examples, a mouse LKB1 gene (both the genomic DNA and cDNA) was cloned; a vector for homologous recombination was constructed using the cloned gene; the vector was introduced into mouse embryonic stem cells (ES cells) to obtain recombinant clones; and the clone was introduced back to an individual mouse which then enabled acquisition of mice having mutations in the LKB1 gene. The present inventors used Cre-loxP system (described later) in creating the recombinant mouse, and thus, achieved phase-specific and tissue-specific induction of mutations in the LKB1 gene. According to this method, the inventors overcame the previous problem of potential embryonic lethality caused by the inactivation of the gene of interest. The mammals in accordance with the present invention (and cell lines established thereof) are expected to be useful as tools to study the onset mechanism of a variety of diseases caused by LKB1 gene deficiency, such as PJS and cancers, and furthermore, are highly useful tools in the development of therapeutic methods and agents for these diseases. Thus, these mammals and cells are expected to be used for various purposes.
The present invention relates to non-human mammals, in which the expression of the LKB1 gene can be or is artificially suppressed, as well as methods for creating the same. More specifically, the present invention relates to the following:
(1) a non-human mammal in which the expression of the endogenous LKB1 gene can be artificially suppressed;
(2) the non-human mammal of (1), wherein the suppression of the expression of the endogenous LKB1 gene is induced by deleting at least a part of the gene or the regulatory region thereof;
(3) the non-human mammal of (1) or (2), wherein at least a part of the LKB1 gene or the regulatory region thereof in the genome is inserted between at least a pair of loxP sequences;
(4) the non-human mammal of any of (1) to (3), wherein the mammal is a rodent;
(5) the non-human mammal of (4), wherein the rodent is a mouse;
(6) a non-human mammal wherein the expression of the endogenous gene encoding LKB1 is artificially suppressed;
(7) the non-human mammal of (6), wherein the expression of the endogenous gene encoding LKB1 is suppressed by a defect in at least a part of the gene or the regulatory region thereof;
(8) the non-human mammal of (6) or (7), wherein the mammal is a rodent;
(9) the non-human mammal of (8), wherein the rodent is a mouse;
(10) a non-human mammalian cell wherein the suppression of the expression of the endogenous gene encoding LKB1 can be artificially induced, further wherein the cell can be differentiated or developed into an individual mammal;
(11) the non-human mammalian cell of (10), wherein the suppression of the expression of the endogenous gene encoding LKB1 is induced by deleting at least a part of the gene or the regulatory region thereof;
(12) the non-human mammalian cell of (10) or (11), wherein at least a part of the LKB1 gene or the regulatory region thereof in the genome is inserted between at least a pair of loxP sequences;
(13) the non-human mammalian cell of (12), wherein the cell contains the Cre gene in an expressible manner;
(14) the non-human mammalian cell of any of (10) to (13), wherein the cell is a rodent cell;
(15) the non-human mammalian cell of (14), wherein the cell is a mouse cell;
(16) the non-human mammalian cell of any of (10) to (15), wherein the cell is an embryonic stem cell;
(17) a non-human mammalian cell, wherein the expression of the endogenous gene encoding LKB1 is artificially suppressed, further wherein the cell can be differentiated or developed into an individual mammal;
(18) the non-human mammalian cell of (17), wherein the expression of the endogenous gene encoding LKB1 is suppressed by a defect in at least a part of the gene or the regulatory region thereof;
(19) the non-human mammalian cell of (18), wherein the cell can be obtained by expressing the Cre gene in the non-human mammalian cell described in (12);
(20) the non-human mammalian cell of any of (17) to (19), wherein the cell is a rodent cell;
(21) the non-human mammalian cell of (20), wherein the cell is a mouse cell;
(22) the non-human mammalian cell of any of (17) to (21), wherein the cell is an embryonic stem cell;
(23) a method for creating the non-human mammal described in any of (1) to (5), which comprises the following steps:
(a) introducing the non-human mammalian cell described in (16) into an embryo obtained from a pregnant female; and
(b) transplanting the embryo into the uterus of a pseudopregnant female;
(24) a method for creating the non-human mammal described in any of (6) to (9), which comprises the following steps:
(a) introducing the non-human mammalian cell described in (22) into an embryo obtained from a pregnant female; and
(b) transplanting the embryo into the uterus of a pseudopregnant female;
(25) a method for creating the non-human mammal described in (7), which comprises the following steps:
(a) providing a fertilized egg or embryo from the non-human mammal described in (3);
(b) expressing the Cre gene in the fertilized egg or embryo after introduction of the gene; and
(c) transplanting the fertilized egg or embryo into the uterus of a pseudopregnant female;
(26) a method for creating the non-human mammal described in (7), which comprises the step to introducing the Cre gene into the non-human mammal described in (3) and expressing the gene; and
(27) a method for creating the non-human mammal described in (7), which comprises the step of mating the non-human mammal described in (3) with a non-human mammal containing a Cre gene in its genome and obtaining their offspring.
According to the present invention, xe2x80x9csuppression of the gene expressionxe2x80x9d includes complete suppression and partial suppression, as well as suppression under specific conditions and also suppression of the expression of either one of the two alleles.
Construction of a Vector for Homologous Recombination (Knockout)
In order to create a knockout animal in which the expression of the LKB1 gene is artificially suppressed, first the LKB1 gene is cloned and then a vector for homologous recombination is constructed by using the gene to inactivate the endogenous LKB1 gene in the target animal.
The vector for homologous recombination contains a nucleic acid sequence designed to inactivate the endogenous LKB1 gene in the target animal. Such a nucleic acid sequence can be, for example, a nucleic acid sequence of the LKB1 gene or the regulatory region thereof containing at least a partial deletion, or alternatively it can be a nucleic acid sequence of the LKB1 gene or the regulatory region thereof containing other genes. A gene which can also function as a marker is preferably selected as the gene to be inserted into the LKB1 gene or the regulatory region thereof. The insert genes to be used include, for example, drug-resistance genes, such as the neomycin-resistance gene (selected by G418 resistance), the thymidine kinase gene (selected by ganciclovir), etc.; toxin genes, such as the diphteria toxin (DT) A gene, etc.; or combinations of these genes. There is no particular limitation on the position where the genes may be inserted in the LKB1 gene, so long as the insertion at that position results in the suppression of the expression of the endogenous LKB1 gene in the target.
Alternatively, the nucleic acid sequence may comprise introns of the LKB1 gene in which the loxP (locus of X-ing-over)sequence derived from phage DNA is inserted at more than 2 sites.
The loxP sequence is a sequence recognized by Cre recombinase (Causes recombination), a site-specific recombination enzyme (Sternberg et al., J. Mol. Biol. 150:467-486, 1981). The Cre recombinase recognizes dual loxP sequences, and catalyzes a site-specific recombination between these sites which results in the removal of the gene located between the loxP sequences (hereinafter abbreviated as Cre-loxP system). The Cre-loxP system used to direct site-specific DNA recombination in transgenic animals is described in Orban et al. (1992) Proc Natl Acad Sci USA. 89(15):6861-5, the contents of which are incorporated herein by reference in their entirety.
An application example that uses this system is shown in FIG. 5. It is possible to prepare embryonic stem cells (ES cells) of the following two types: (1) the cell containing conventional gene deletion (type 1), and (2) cell having conditional gene deletion (type 2), by constructing a vector having 3 loxP sequences for homologous recombination, then introducing the vector, for example, into ES cells to obtain recombinant cells, and allowing for the transient expression of the Cre recombinase in the resulting ES cells. When a construct with 3 loxP sequences is used, two types of ES cell clones, each having distinct a genotype, can be advantageously prepared from a single type of recombinant ES cell merely by expressing the Cre recombinase. An individual derived from the type-2 ES cell exhibits the wild-type phenotype, but it can be converted to type 1 by expressing the Cre recombinase gene.
Instead of the Cre-loxP system, it is also possible to use the combination of the FRT (Flp recombinase target) sequence and Flp recombinase from yeast which recognizes the sequence for site-specific recombination.
The insertion of these genes into the cloned LKB1 gene can be carried out in vitro by using conventional DNA recombination techniques (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).
Transfection of the Cell
The homologous recombination vector constructed as above is introduced into a non-human mammalian cell capable of differentiating (or developing) into an individual mammal (for example, an ES cell) for homologous recombination with the LKB1 gene in the cell. There is no particular limitation on the type of the organism from which the non-human mammalian cell to be used in the present invention is derived, but preferably it is a rodent such as mouse, rat, hamster, rabbit, and so on.
The introduction of the homologous recombination vector into cells can be performed by methods well known to those skilled in the art, for example, the electroporation method. The introduction results in recombination between the cellular LKB1 gene and the corresponding region of the homologous recombination vector in some population of the cells. In this manner, the wild-type gene is substituted with the gene having the genotype constructed in the homologous recombination vector. Thus, it is possible to obtain cells having an LKB1 gene in which a marker gene and/or loxP sequences have been introduced.
When the homologous recombination vector contains a marker gene, cells can be selected according to this marker gene as an index, due to the loss of the LKB1 gene and the acquired marker gene at the same time in cells where desired homologous recombination has taken place. For example, when a drug-resistance gene is used as the marker gene, cells in which the desired homologous recombination has taken place can be selected by culturing the cells in the presence of the drug at a lethal level subsequent to the introduction of vectors,
However, when the Cre-loxP system is used, in some cases, the vector for homologous recombination is designed so that the loxP sequence and the marker gene are integrated into the introns, and accordingly, the LKB1 gene is not always inactivated in the cells. In this system, inactivation of the LKB1 gene can be achieved by expressing the Cre recombinase in the cells to remove the region inserted between a pair of loxP sequences.
The Cre recombinase in the cells can be expressed, for example, by methods employing expression vectors such as adenoviral vectors, or alternatively by mating transgenic animals in which the expression of Cre is regulated by a promoter capable of regulating the expression in a tissue-specific or phase-specific manner with mammals having the Cre-loxP system. The first mating of a transgenic animal having regulated Cre expression with another animal having the Cre-loxP system, results in knockout newborns in which only one allele of the LKB1 gene is deleted (heterozygote), but by further mating these animals, knockout animals in which both alleles of the LKB1 gene are deleted can be obtained.
Injection into Embryos and Transplantation of Embryos
When ES cells are used in the present invention, the cells are injected into blastocysts to prepare chimera embryos. Further, the chimera embryos are transferred into the horn of uterus of pseudopregnant mammals to obtain newborns. The blastocysts to be used for the injection can be obtained by perfusing the uterus of a pregnant female. To determine whether or not the ES cell has been incorporated in the developing embryo after the creation of an individual mammal, it is preferable to select the type of blastocyst that gives different external characteristics (for example, fur color) to distinguish the origin of a cell, whether it is derived from the ES cell or blastocyst, in the created animal.
Subsequently, newborns are obtained by mating the resulting chimera animal with an animal of an appropriate strain of the same species. When the germline of the chimera animal is derived from the homologous recombinant ES cell, it is possible to obtain newborns in which the LKB1 gene has been deleted. However, when the Cre-loxP system is used, some of the homologous recombination vectors may be designed to integrate the loxP sequence and the marker gene into an intron. In this case, the LKB1 gene is not always inactivated. In such cases, it is possible to inactivate the LKB1 gene by expressing the Cre recombinase in the somatic cell or germline cell, such as fertilized egg.
When somatic cells other than ES cells are used in the present invention, it is possible to create a knockout animal by using techniques for creating somatic cell cloned animals. Specifically, for example:
1) a cell that contains a gene to which a mutation is introduced through the homologous recombination is established by the same method as that used for ES cells, using cells other than ES cells such as fibroblast cells;
2) an animal carrying the mutated gene is created from this cell by using the method for creating somatic cell cloned animals (Wilmut et al., Nature 385:810-803, 1997; Wakayama et al., Nature 394:369-374, 1998).
3) the resulting animal newborn carrying the mutated gene corresponds to the F1 mouse of the method using the ES cell, and can be used thereafter according to a same manner as the ES cell.
Use of the Knockout Animal
Knockout animals of the present invention are useful for developing therapeutics and methods to treat a variety of diseases caused by functional defects of the LKB1 gene. For example, a test compound is administered to the knockout animals of the present invention, and the influences on polyp formation, carcinogenesis, and pigment macule formation are tested to select compounds exhibiting desired effects.
Furthermore, cells prepared from knockout animals can be used for developing therapeutics or treatment methods. For example, cells are prepared from embryos and such from knockout animals of the present invention, and then a test compound is added to the cells to determine the influence on cell proliferation, ability to form colony in soft agar or the like, focus forming ability, cell motility, and such, and thereby selecting compounds exhibiting the desired effect. The cells may be primary culture cells or established cell lines. The compounds screened are candidates for pharmaceutical agents.